Saturating mutagenesis with assembled CRISPR-Cas9 ribonucleoprotein complexes.

ABSTRACT: The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Danio rerio

SUBMITTER: Mark Robinson 

PROVIDER: E-MTAB-4143 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress